There is a need for standardized validation methods for antibody specificity and selectivity. A recent article published in Nature Communications by Edfors et al. shows that the five pillars proposed by the International Working Group for Antibody Validation (IWGAV) can be used to validate antibodies for Western Blot applications in a standardized and systematic manner.

There is a need for standardized validation methods for antibody specificity and selectivity. A recent article published in Nature Communications by Edfors et al. shows that the five pillars proposed by the International Working Group for Antibody Validation (IWGAV) (Uhlén et al., Nature Methods 2016) can be used to validate antibodies for Western Blot applications in a standardized and systematic manner. These validation methods include orthogonal methods, genetic knockdown, recombinant expression, independent antibodies and capture mass spectrometry analysis. The authors show that more than 6,000 antibodies published on the Human Protein Atlas could be validated in this enhanced manner using at least one of the five strategies presented in their article, and more than 1,600 of these antibodies were validated by at least two of the pillars and 267 with three or more pillars.

One of the main focuses in this work has been to introduce orthogonal methods for validation of antibodies. This strategy opens up streamlined efforts to validate antibodies using verified and standardized cell or tissue lysate panels centrally obtained by certified providers, in which the antibody-independent method is performed and the data used for validation is provided and shared in an open access manner.

The authors also show that all the validation strategies described here can be used to investigate and validate antibodies that yield several bands on the Western blot assay. This phenomenon is not uncommon and could either be due to unspecific staining as a result of off-target binding or specific staining of multiple isoforms of the target protein due to proteolysis, post-translational modifications or splice variants. This includes identification and label free quantification of proteins and their migration in the gel for 5,605 human target proteins. The complete work can be accessed through the link https://www.nature.com/articles/s41467-018-06642-y.

More information about the on-going validation effort currently carried out within the Human Protein Atlas was presented by Professor Mathias Uhlén during the 3rd International Antibody Validation Meeting in Bath, September 20, 2018.

To watch the lecture go to https://www.youtube.com/watch?v=W1_Bv0Iu-so&feature=youtu.be

Publication: Edfors F, Hober A, Linderbäck K, Maddalo G, Azimi A, Sivertsson Å, Tegel H, Hober S, Szigyarto CA, Fagerberg L, von Feilitzen K, Oksvold P, Lindskog C, Forsström B, Uhlen M.
Enhanced validation of antibodies for research applications.
Nat Commun. 2018 9(1):4130.

Fredrik Edfors